CN111035807B

CN111035807B - A kind of preparation method of ultra-thin APCS graft

Info

Publication number: CN111035807B
Application number: CN202010000241.1A
Authority: CN
Inventors: 张莹莹; 刘欣; 胡芷馨; 张明昌; 谢华桃; 刘书婷; 陈华; 彭曦
Original assignee: Union Hospital Tongji Medical College Huazhong University of Science and Technology
Current assignee: Union Hospital Tongji Medical College Huazhong University of Science and Technology
Priority date: 2020-01-02
Filing date: 2020-01-02
Publication date: 2022-02-11
Anticipated expiration: 2040-01-02
Also published as: CN111035807A

Abstract

The invention discloses a preparation method of an ultrathin APCS (apo-optic computed tomography) implant, which relates to the field of corneal endothelium, and comprises the steps of cleaning and dehydrating a acellular pig corneal stroma, precooling a freezing microtome to a temperature lower than 20 ℃, smearing an OCT (optical coherence tomography) embedding medium on a sample holder, flattening, placing the dehydrated stroma on the OCT embedding medium, freezing, setting the slice thickness of the freezing microtome to be 20-60 mu m, and cutting to obtain the ultrathin APCS implant. The ultrathin APCS implant with the thickness of 20-60 mu m is obtained, the fusion of the implant and the eyes of a patient is effectively improved, the success rate of an operation is improved, meanwhile, the cost of the freezing microtome is low, the operation method is simple, the preparation cost of the implant can be effectively reduced, and the medical cost of the patient is reduced.

Description

Preparation method of ultrathin APCS (amorphous silicon copper-zinc copper) implant

Technical Field

The invention relates to the field of preparation of corneal endothelium, in particular to a preparation method of an ultrathin APCS (amorphous silicon copper calcium copper sulfate) implant.

Background

The corneal endothelium maintains the transparency and thickness of the cornea due to the barrier effect formed by the tight connection among the cells and the sodium-potassium pump function, the visual protection effect is vital, the corneal endothelial cells can not be regenerated in vivo basically, the density of the corneal endothelial cells can be obviously reduced in trauma or surgical trauma, refractive surgery, early penetrating or endothelial corneal transplantation, endothelial malnutrition or certain disease stresses (such as diabetes or glaucoma), and once the density of the corneal endothelial cells is lower than 400-500 cells/mm²In time, interstitial edema, corneal opacity, bullous keratopathy and visual deterioration cascade reactions occur.

With the aging of the population, the incidence of diabetes, glaucoma and cataract is gradually increased, cataract surgery is more and more common, endothelial injury is a common complication of the cataract, the endothelial injury can cause blindness of patients, and the surgery mainly depends on corneal transplantation and DMEK/DSAEK surgery (DMEK, Descemet membrane endothial keratoplasty, DSAEK and DSAEK surgery), but the corneal transplantation and the DMEK/DSAEK surgery both depend on corneas donated by other people, and most patients are difficult to be treated in time due to the extremely limited number of donors.

The development of the corneal tissue engineering technology makes it possible to reconstruct the tissue engineering human cornea in vitro, the tissue engineering human cornea stroma reconstructed in vitro is used as a substitute for donated cornea and has become a clear hope for a plurality of patients with corneal blindness through cornea transplantation, human corneal endothelial cells are cultured in vitro, and the tissue functional cornea is constructed by being planted on various biological materials simulating the human corneal elastic layer and the stroma in vivo for transplantation, wherein the acellular porcine cornea stroma is used as a substitute for the human cornea stroma for clinical tests, the tissue compatibility, the immunogenicity and the safety of the acellular porcine cornea stroma are tested, and the tissue engineering technology is a mature biological material which is widely applied by the existing corneal tissue engineering technology.

At present, before the porcine corneal stroma is used, the porcine corneal stroma is generally required to be cut into a graft for use, and currently, a femtosecond laser is generally used for cutting: the porcine cornea is firstly cut into the corneal endothelial sheet with the thickness of not less than 100 mu m, and then the corneal endothelial sheet is decellularized for standby, however, the cost of the corneal endothelial sheet is higher and the medical cost of a patient is increased due to higher price and more complex operation of a femtosecond laser instrument, and meanwhile, the corneal endothelial sheet with the thickness of not less than 100 mu m is poorer in fusion with the ocular cells of the patient after the operation due to thicker thickness, and the operation is easy to fail.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a preparation method of an ultrathin APCS implant, which can reduce the cost of corneal endothelial cells and improve the success rate of surgery on the basis of reducing the thickness of the corneal endothelial cells.

In order to achieve the above purposes, the technical scheme adopted by the invention is as follows:

a preparation method of an ultrathin APCS implant comprises the following steps:

cleaning and dehydrating the acellular pig cornea matrix, precooling a freezing microtome to be lower than 20 ℃, smearing an OCT embedding medium on a sample holder, flattening, placing the dehydrated matrix on the OCT embedding medium, freezing, setting the slice thickness of the freezing microtome to be 20-60 mu m, and cutting to obtain the ultrathin APCS implant.

Further, the cleaning and dehydration of the acellular porcine corneal stroma specifically comprises the following steps: and (3) absorbing glycerol on the surface of the acellular porcine cornea matrix, putting the acellular porcine cornea matrix into a culture dish with PBS or normal saline for hydration treatment until the matrix is completely edematous and is adhered and loosened with the bottom of the culture dish, cleaning the edematous matrix until no glycerol exists, putting the matrix into a hypertonic solution for dehydration until the matrix is settled to the bottom of the hypertonic solution.

Further, the hypertonic solution comprises 3g of sucrose and 7ml of PBS, and the concentration of the hypertonic solution is 30%.

Further, the step of smearing the OCT embedding medium on the sample holder and flattening specifically comprises the following steps: and smearing an OCT embedding medium on the sample support and precooling, clamping the sample support on a freezing microtome after the OCT embedding medium is completely solidified, setting the thickness of a slice of the freezing microtome to be 30-50 mu m, starting a coarse advance and retreat key, and rotating a knob to flatten the OCT embedding medium.

Further, the diameter of the solidified OCT embedding medium is larger than that of the acellular porcine corneal stroma.

Further, the step of placing the dehydrated matrix on an OCT embedding medium and freezing, setting the slice thickness of a freezing microtome to be 20-60 mu m and cutting to obtain the ultrathin APCS implant specifically comprises the following steps: placing the dehydrated matrix on an OCT embedding medium, smearing the OCT embedding medium on the matrix until the matrix is embedded, placing a sample holder with the matrix on a freezing frame until the OCT embedding medium on the surface of the matrix is completely frozen, marking the bottom edge of the matrix, smearing the OCT embedding medium on the surface of the matrix once and freezing, firstly setting the thickness of a section of a freezing microtome to be 30-50 mu m, starting a coarse advance and retreat key, rotating a knob until the mark on the bottom edge of the matrix is positioned in a cutting visual field, then setting the thickness of the section of the freezing microtome to be 20-60 mu m, and cutting to obtain the corresponding ultrathin APCS implant.

Further, the method for obtaining the corresponding ultra-thin APCS implant further comprises the following steps: endothelial cells are planted on the ultrathin APCS plant until the cells are fused, and the transplantable endothelial cell plant is obtained.

Further, the planting of endothelial cells into cell fusion in the ultra-thin APCS plants comprises the following steps: cleaning and sterilizing the surface of the ultra-thin APCS implant, putting the ultra-thin APCS implant into a cell culture hole, injecting endothelial cell suspension into the cell culture hole, and culturing until cells are fused.

Further, the cleaning and sterilizing treatment of the surface of the ultra-thin APCS implant comprises the following steps: placing the ultrathin APCS implant into a culture dish filled with PBS, flushing the implant until the surface of the implant is free of OCT embedding agent, soaking the ultrathin APCS implant for 30min by using the PBS with double antibodies, placing the implant into a culture hole with PBS solution, slowly absorbing the PBS to enable the implant to be smoothly attached to the bottom of a culture plate, placing the culture plate into a super clean bench, and opening an ultraviolet lamp to irradiate for 30min for sterilization.

An ultra-thin APCS implant, the thickness of the implant is 20-60 μm.

Compared with the prior art, the invention has the advantages that:

(1) according to the preparation method of the ultrathin APCS implant, the acellular porcine cornea matrix is cut by a cold freezing microtome to obtain the ultrathin APCS implant with the thickness of 20-60 microns, the thickness of the implant is far smaller than that of the implant cut by a femtosecond laser instrument, the fusion of the implant and the eyes of a patient can be effectively improved, the success rate of an operation is improved, meanwhile, the cost of the freezing microtome is low, the operation method is simple, the preparation cost of the implant can be effectively reduced, and the medical cost of the patient is reduced.

Detailed Description

The following examples of the present invention are described in further detail.

The embodiment of the invention provides a preparation method of an ultrathin APCS (porcine corneal stroma) implant, which comprises the following steps: cleaning and dehydrating a acellular pig cornea matrix, precooling a freezing microtome to be lower than 20 ℃, coating an OCT embedding medium (a water-soluble mixture of polyethylene glycol and polyvinyl alcohol) on a sample holder, flattening, placing the dehydrated matrix on the OCT embedding medium, freezing, setting the slice thickness of the freezing microtome to be 20-60 mu m, and cutting to obtain the ultrathin APCS (advanced persistent personal computer) implant.

The method specifically comprises the following steps:

s1, absorbing glycerol on the surface of the acellular pig cornea stroma, putting the acellular pig cornea stroma into a culture dish with PBS (phosphate buffer saline) or normal saline for hydration treatment until the stroma is completely edematous and the adhesion of the stroma to the bottom of the culture dish is loosened, washing the edematous stroma until no glycerol exists, putting the stroma into a hypertonic solution, and dehydrating the stroma overnight at 4 ℃ until the stroma is settled to the bottom of the hypertonic solution, wherein the concentration of the hypertonic solution is 30%, and the hypertonic solution comprises 3g of sucrose and 7ml of PBS.

S2, pre-cooling the freezing microtome to a temperature lower than 20 ℃, smearing an OCT embedding medium on the sample support, pre-cooling (the smearing area of the OCT embedding medium is large to ensure that the diameter of the solidified OCT embedding medium is larger than that of a acellular pig cornea matrix), clamping the sample support on the freezing microtome after the OCT embedding medium is completely solidified, setting the thickness of a slice of the freezing microtome to be 30-50 mu m, starting a coarse advance and retreat key, and rotating a knob to flatten the OCT embedding medium.

S3, placing the dehydrated matrix on the surface of the modified OCT embedding medium, flattening the dehydrated matrix as much as possible, smearing the OCT embedding medium on the matrix until the matrix is embedded, placing the sample support with the matrix on a freezing frame until the OCT embedding medium on the surface of the matrix is completely frozen, marking the bottom edge of the matrix, smearing the OCT embedding medium on the surface of the matrix once and freezing, firstly setting the slice thickness of a freezing microtome to be 30-50 mu m, starting a coarse advance and retreat key, rotating a knob until the mark on the bottom edge of the matrix is positioned in a cutting visual field, adjusting the position and the angle of a rolling-proof plate to a proper position according to actual conditions, then setting the slice thickness of the freezing microtome to be 20-60 mu m, and cutting to obtain the corresponding ultrathin APCS implant.

S4, placing the ultrathin APCS implant into a culture dish filled with PBS, flushing the implant until the surface of the implant is free of OCT embedding medium, soaking the ultrathin APCS implant for 30min by using PBS with double antibodies, placing the implant into a culture hole with PBS solution, slowly absorbing the PBS to enable the implant to be smoothly attached to the bottom of a culture plate, placing the culture plate into a super clean bench, and opening an ultraviolet lamp to irradiate for 30min for sterilization.

S5, placing the sterilized ultrathin APCS implant into cell culture holes, injecting 0.5-1 ml of endothelial cell suspension into each cell culture hole, gently shaking to uniformly distribute cells, centrifuging the culture plate for 5-10 minutes at the rotating speed of 800-1200 rpm to promote cell adhesion, then standing for culture, replacing the culture medium every 2-3 days, observing the cell state, and obtaining the transplantable endothelial cell implant after cell fusion.

According to the invention, the acellular pig cornea matrix is cut by the freezing microtome to obtain the ultrathin APCS (amorphous silicon copper-copper) implant with the thickness of 20-60 microns, the thickness of the implant is far smaller than that of the implant cut by a femtosecond laser instrument (not smaller than 100 microns), the fusion of the implant and the eye of a patient can be effectively improved, the success rate of an operation is improved, meanwhile, the cost of the freezing microtome is lower, the operation method is simpler, the preparation cost of the implant can be effectively reduced, and the medical cost of the patient is reduced.

The invention also provides the ultrathin APCS implant prepared based on the method, the thickness of the implant is 20-60 mu m, and endothelial cells are fused on the surface of the implant.

The present invention is not limited to the above-mentioned preferred embodiments, and any other products in various forms can be obtained by anyone with the teaching of the present invention, but any changes in the shape or structure thereof, which have the same or similar technical solutions as the present invention, are within the protection scope.

Claims

1. a preparation method of ultra-thin APCS implant, is characterized in that: comprise the following steps:

The decellularized porcine corneal stroma was cleaned and dehydrated, the cryostat was pre-cooled to below 20°C, the OCT embedding medium was smeared on the sample holder and smoothed, and the dehydrated matrix was placed on the OCT embedding medium and frozen. Setting the slice thickness of the cryostat to be 20-60 μm and cutting to obtain ultrathin APCS grafts;

The cleaning and dehydration of the decellularized porcine corneal stroma specifically includes the following steps: sucking the glycerol on the surface of the decellularized porcine corneal stroma, putting it into a petri dish with PBS or normal saline, and performing hydration treatment until the stroma is completely edematous and mixed with the petri dish. The adhesion of the bottom is released, and the edema matrix is cleaned to no glycerin, and then placed in a hypertonic solution for dehydration until the matrix settles to the bottom of the hypertonic solution;

The smearing and smoothing of the OCT embedding agent on the sample holder specifically includes the following steps: smearing the OCT embedding agent on the sample holder and pre-cooling, after the OCT embedding agent is completely solidified, the sample holder is clamped on the cryostat, Set the slice thickness of the cryostat to 30-50 μm, activate the coarse forward and backward keys, and turn the knob to level the OCT embedding medium;

The dehydrated matrix is placed on the OCT embedding medium and frozen, and the slice thickness of the cryostat is set at 20-60 μm and cut to obtain an ultra-thin APCS graft, which specifically includes the following steps: placing the dehydrated matrix on the On the OCT embedding medium and apply the OCT embedding medium on the matrix until the matrix is embedded, place the sample holder with the matrix on the freezing rack until the OCT embedding medium on the surface of the matrix is completely frozen, and mark the bottom edge of the matrix. , apply OCT embedding medium on the surface of the matrix once and freeze, first set the slice thickness of the cryostat to 30-50 μm, start the coarse advance and retreat key, turn the knob until the mark on the bottom edge of the matrix is in the cutting field of view, and then set the cryostat The thickness of the slices is 20-60 μm and cut to obtain the corresponding ultra-thin APCS grafts;

After obtaining the corresponding ultra-thin APCS graft, the method further includes the following steps: planting endothelial cells on the ultra-thin APCS graft until the cells are fused to obtain a transplantable endothelial cell graft.

2 . The method for preparing an ultra-thin APCS graft according to claim 1 , wherein the hypertonic solution comprises 3 g of sucrose and 7 ml of PBS, and the concentration of the hypertonic solution is 30%. 3 .

3. the preparation method of a kind of ultrathin APCS graft as claimed in claim 2, is characterized in that: described in the ultrathin APCS graft planting endothelial cell to cell fusion comprises the following steps: carry out the ultrathin APCS graft surface After cleaning and sterilization, the ultrathin APCS explants were placed in the cell culture wells, and the endothelial cell suspension was injected into the cell culture wells and cultured until the cells were confluent.

4. the preparation method of a kind of ultra-thin APCS implant as claimed in claim 3, is characterized in that: the described ultra-thin APCS implant surface is cleaned and sterilized comprises the following steps: the ultra-thin APCS implant is placed Put it into a petri dish with PBS, rinse the graft until there is no OCT embedding agent on the surface, soak the ultra-thin APCS graft in PBS with double antibody for 30 minutes, put it into the culture hole with PBS solution, and slowly suck it up. In addition to PBS, the grafts were flatly attached to the bottom of the culture plate, and the culture plate was placed in the ultra-clean bench, and the UV lamp was turned on for 30 minutes to sterilize.

5 . An ultra-thin APCS implant prepared by the method according to any one of claims 1 to 4 , wherein the thickness of the implant is 20-60 μm. 6 .